Age-dependent upregulation of Y RNAs in Caenorhabditis elegans

Y RNA is a conserved small non-coding RNA whose functions in aging remain unknown. Here, we sought to determine the role of C. elegans Y RNA homologs, CeY RNA (CeY) and stem-bulge RNAs (sbRNAs), in aging. We found that the levels of CeY and sbRNAs generally increased during aging. We showed that CeY was downregulated by oxidative and thermal stresses, whereas several sbRNAs were upregulated by oxidative stress. We did not observe lifespan phenotypes by mutations in CeY-coding yrn-1. Future research under various genetic and environmental conditions is required to further evaluate the role of Y RNA in C. elegans aging.


Description
Various non-coding RNAs (ncRNAs) change their levels during aging, and influence lifespan in many species, including Caenorhabditis elegans (Garg and Cohen 2014;He et al. 2018;Lee 2019, Kim et al. 2021). However, the role of Y RNAs, ncRNAs that regulate DNA replication and RNA quality control , in aging and lifespan remains unknown. C. elegans genome encodes 19 Y RNA homologs, C. elegans Y RNA (CeY) encoded by yrn-1 and 18 stem-bulge RNAs (sbRNAs), each of which contains a conserved stem-loop structure ( Fig. 1A) . Y RNAs form a Ro60 ribonucleoprotein by binding Ro 60-kDa protein (Ro60), which is conserved from bacteria to human (Sim et al. 2020). ROP-1, the C. elegans Ro60, binds and stabilizes CeY (Van Horn et al. 1995;Labbé et al. 1999). rop-1 mRNA and ROP-1 protein levels increase during aging in C. elegans (Liang et al. 2014;Mansfeld et al. 2015;Angeles-Albores et al. 2017), suggesting the potential aging-regulatory functions of ROP-1 and its associated RNA, CeY. Here, we sought to determine the role of the Y RNA homologs in aging and lifespan.
We first tested whether the levels of C. elegans Y RNA homologs changed during aging by performing real-time quantitative reverse transcription PCR (qRT-PCR) analysis. We found that the levels of the CeY and sbRNAs were generally increased during aging (Fig. 1B). In addition, we showed that the mRNA level of rop-1 was increased during aging ( Increased stress resistance is associated with longevity in multiple species (Epel andLithgow 2014, Park et al. 2017), and stress-responsive factors play crucial roles in aging (Son et al. 2019). We therefore measured the effect of oxidative or thermal stresses on the levels of C. elegans Y RNA homologs. We found that the level of CeY was decreased under both oxidative ( Fig. 1C) and thermal stress conditions (Fig. 1D). We also showed that oxidative stress significantly increased the levels of four sbRNAs, Ce4, Ce5, CeN75, and CeN73-1 (Fig. 1C), whereas thermal stress did not ( Fig. 1D; see Figure 1D legend for discussion). In contrast to the decrease in the level of CeY under these tested stress conditions, the mRNA level of rop-1 was increased by oxidative stress (Fig. 1C) but not by thermal stress (Fig. 1D). Thus, it seems likely that these two external stresses affect the expression of various C. elegans Y RNA homologs and rop-1 differently.
We then focused our functional analysis on CeY by using CRISPR/Cas9 gene targeting, because the potential functional redundancy of 18 sbRNAs may hinder characterizing the roles of individual sbRNAs in aging. We generated two mutant alleles, yh84 and yh85, in yrn-1 gene that encodes CeY (Fig. 1E). We showed that yh84 or yh85 mutation substantially reduced the level of CeY (Fig. 1F). In addition to these yrn-1 mutants, we characterized two different rop-1 mutant alleles, rop-1(pk93) and rop-1(ok2690), both of which substantially reduced the level of CeY and rop-1 mRNA (Fig. 1F, G) (Labbé et al. 1999). We then measured the lifespan of these mutants, and found that yrn-1(yh84) or yrn-1(yh85) mutation did not affect the lifespan of wild-type animals at 20°C (Fig. 1H, I, M, N). Reducing insulin/IGF-1 signaling via inhibition of daf-2/insulin/IGF-1 receptor doubles lifespan (Kenyon et al. 1993), and is one of the most studied longevity-promoting interventions in C. elegans (Altintas et al. 2016). A major function of insulin/IGF-1 signaling in C. elegans is to regulate the proper formation of dauer, an alternative hibernation-like developmental stage (Hu 2007). rop-1(pk93) mutations decrease dauer formation in a wild-type background, and enhance or suppress the increased dauer formation by daf-2(-) mutants in an allele-specific manner (Labbé et al. 2000). These findings raise the possibility that rop-1 and/or CeY affects physiological processes regulated by insulin/IGF-1 signaling. However, yrn-1(yh84) or yrn-1(yh85) mutation had little effect on the lifespan of daf-2 RNAi-treated worms (Fig.  1H, I, M, N). Therefore, CeY appears to be dispensable for maintaining normal lifespan and promoting longevity by reduced insulin/IGF-1 signaling. We also found that rop-1(pk93) mutations decreased the lifespan of both control RNAi-and daf-2 RNAi-treated worms (Fig. 1J, M, N), whereas rop-1(ok2690) mutation did not (Fig. 1K, M, N). A previous study reported that rop-1(pk93) mutations do not affect lifespan (Labbé et al. 1999). We found that rop-1(pk93) mutations substantially increased vulval rupturing during adulthood (Fig. 1L), which reduces lifespan (Leiser et al. 2016). Thus, mutations in rop-1 do not appear to directly affect aging per se but may cause early deaths during adulthood by increasing vulval rupture phenotypes in our experimental conditions. Together, we did not find apparent lifespan phenotypes in yrn-1 or rop-1 mutants, both of which displayed substantially reduced CeY levels.
Our study points to the possibility that CeY may not directly participate in the regulation of lifespan. CeY alone may not be functionally equivalent to the vertebrate Y RNA, as CeY does not affect DNA replication, likely because of the lack of conserved motif in the upper stem (Gardiner et al. 2009;Boria et al. 2010;. In contrast, sbRNAs contain a conserved UG-CA motif (Fig. 1A) and participate in DNA replication . Thus, it will be important to test whether the inhibition of sbRNAs affects lifespan. In particular, investigating the roles of four oxidative stress-induced sbRNAs, Ce4, Ce5, CeN75, and CeN73-1, which were also age-dependently upregulated, in aging and lifespan will be crucial for future research.

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qRT-PCR analysis. qRT-PCR analysis was performed as previously described (Park et al. 2020). For qRT-PCR analysis using aged worms, wild-type (N2) animals were synchronized at L1 stage by embryo bleaching (Stiernagle 2006) followed by incubation in S-basal medium at 20°C with a gentle rotation for 16 h. Approximately 4,800 to 9,000 synchronized L1 animals were placed and cultured on nematode growth medium (NGM) plates seeded with E. coli OP50. L4 larvae were then treated with 50 µM 5-fluoro-2′-deoxyuridine (FUDR; Sigma-Aldrich, MO, USA) to prevent progeny from hatching. Fertile adult animals at days 1, 4, 7, or 11 were harvested and used for total RNA extraction. Total RNA was extracted by using RNAiso plus (Takara, Shiga, Japan), and cDNA was synthesized by using ImProm-II Reverse Transcriptase (Promega, WI, USA) following manufacturer's instruction. Real-time quantitative PCR was performed using Power SYBR Green PCR master mix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) and StepOne real-time PCR system (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Relative quantity was calculated by comparative C T method using pmp-3 and small nucleolar RNA sn2343 as endogenous controls for rop-1 mRNA and CeY/sbRNAs, respectively. P values were calculated by two-tailed Student's t test. For qRT-PCR analysis using animals under oxidative or thermal stress conditions, approximately 5,600 stage-synchronized wild-type animals were grown on NGM plates seeded with OP50 at 20°C until they reached L4 stage. The L4 larvae were then exposed to 7.5 mM tert-butyl hydroperoxide (t-BOOH) for 4 h for eliciting oxidative stress (Goh et al. 2018) or placed in a 33°C incubator for 30 min for thermal stress (Brunquell et al. 2016). The animals were then harvested and washed three times with M9 buffer, and stored at -80°C before total RNA extraction and qRT-PCR analysis. A heat map showing age-dependent changes in levels of Y RNAs and rop-1 mRNA was generated by Heatmapper (http://www.heatmapper.ca) (Babicki et al. 2016). For qRT-PCR analysis of CeY or rop-1 mRNA using yrn-1 (IJ1991 and IJ1992) and rop-1 mutants (MQ470 and RB2032), approximately 6,000 to 8,000 bleach-synchronized animals were grown until developing to prefertile adults. The animals were then harvested and washed three times with M9 buffer, and stored at -80°C before extracting total RNA for qRT-PCR analysis.

Wild-type: ttaaacatttGGGCTCGGTCCGAGTTTCATGGTCTCCAATGTGTGTGTGTGTGTGTTTTCTTTAGGAACC TCGGTTCCAACCTCATCTTGACCTTGAAACTACTTTGACCGCTCCttttggattt
yrn-1(yh84): ttaaataggagaaataagacactaagacaataagagaaatattttGGGCTCGGTCCGAGTTTCATGGTCT CCAATGTGTGTGTGTGTGTGTTTTCTTTAGGAACCTCGGTTCCAACCTCATCTTGACCTTGAAACTACTT TGACCGCTCCttttggattt yrn-1(yh85): ttaaacatttGGCTCGGTCCGAGTTTCATGGTCTCCAATGTGTGTGTGTGTGTGTTTTCTTTAGGAACC ccattt Lifespan analysis. Lifespan assays were performed as previously described with some modifications . Plates for feeding RNAi induction were prepared by growing HT115 RNAi bacteria containing empty vector (pAD12; control RNAi) or daf-2 double-stranded RNA-expressing vector (pAD48; daf-2 RNAi) on NGM plates containing ampicillin (50 μg/ml; USB, Santa Clara, CA, USA) and isopropyl-β-D-thiogalactoside (1 mM; Gold Biotechnology, St. Louis, MO, USA) at 37°C. Stagesynchronized wild-type, outcrossed yrn-1(yh84) and yrn-1(yh85) mutants, and rop-1(pk93) and rop-1(ok2690) mutants were placed on the plates and fed with control or daf-2 RNAi bacteria at 20°C until reaching a prefertile adult stage. The animals were then transferred to new plates containing 5 µM FUDR. Animals that did not respond to a gentle touch using a platinum wire were counted as dead. Animals that ruptured, bagged, crawled off or burrowed the plates were censored but included in the statistical analysis. Lifespan assays were performed as duplicates by two independent researchers double-blindly. The lifespan data from two replicates were pooled to generate lifespan curves shown in Fig. 1H-K, but the statistical analysis was performed separately and shown in Fig. 1M and 1N. Statistical analysis of lifespan data was performed using OASIS2 (http://sbi.postech.ac.kr/oasis2) (Han et al. 2016), and P values were calculated by Log-rank (Mantel-Cox) test.